In vitro embryo production (IVP), which comprises in vitro oocyte maturation (IVM), in vitro fertilization (IVF) and in vitro embryo culture (IVC), holds many benefits in agricultural livestock settings, facilitating the mass production of embryos at low cost. From 2010 to 2012, the amount of transferred IVP bovine embryos has been raised over eight times, reaching 350,000 annually. Moreover, commercial bovine IVP embryo transfer has become a large international business to reduce costs of genetic export and import (2013). However, the existing methods for IVM of bovine oocytes, always lead to nuclear-cytoplasmic asynchrony, which would compromise cytoplasmic maturation of oocytes and subsequent embryonic development. Therefore, nuclear-cytoplasmic asynchrony of IVM oocyte is the primary bottleneck for the large-scale application of IVP process in enhancing bovine reproductive efficiency.
Efficient IVP process is largely dependent on the fully matured oocytes. The oocytes used for IVM were mainly collected from ovaries by ovum pick-up (OPU) or from slaughtered animals. Distinct with naturally (in vivo) matured oocytes, the oocytes used for IVM are recovered from 3-8 mm follicles. In these follicles, immature oocytes are maintained at meiotic arrest, which is also referred as germinal vesicle (GV) stage. The isolation of GV oocyte from its follicular environment triggers meiotic resumption (also referred to as “spontaneous nuclear maturation”). During the process of conventional IVM, spontaneous nuclear maturation causes a premature breakdown of oocyte-cumulus cell gap junctions (Thomas et al., 2004), which impairs the intercellular transport of beneficial cumulus cell metabolites to oocytes (Gilchrist and Thompson, 2007). Therefore, the conventional IVM systems always lead to an insufficient cytoplasmic maturation in large part of oocytes that nuclear maturation are fully achieved. This is referred as “nuclear-cytoplasmic asynchrony”.
Numerous efforts have been made to overcome the nuclear-cytoplasmic asynchrony of IVM oocytes. Many chemical agents that could inhibit oocyte meiotic resumption, were used to inhibit spontaneous maturation temporarily and thus improve the synchronization between nuclear and cytoplasmic maturation, which could enhance developmental competence of oocytes. These chemicals, such as roscovitine, butyrolactone (Mermillod et al., 2000), cycloheximide (Kastrop et al., 1991), 6-DMAP (Saeki et al., 1997), forskolin (Albuz et al., 2010), etc., always showed evident inhibitory effect on nuclear maturation. Furthermore, some of those meiotic inhibitors could improve subsequent developmental rate of preimplantation embryos. However, the safety of these agents for oocytes and embryos has not been fully evaluated and certain of agents have been identified to have detrimental effects on oocyte ultrastructure and function (Fair et al., 2002; Lonergan et al., 2003). Furthermore, inappropriate usage of chemical agents during oocyte IVM could subsequently impair implantation and fetal development after embryo transfer (Albuz et al., 2010).
Therefore, safe, efficient and reversible oocyte meiotic inhibition during IVM is critical for oocyte cytoplasmic maturation and subsequent embryonic development. It would be desirable if endogenous physiological factors responsible for oocyte meiotic arrest could be used for inhibiting spontaneous meiotic resumption, thereby improving nuclear-cytoplasmic synchronization and subsequent developmental competence of IVM oocytes.
C-type natriuretic peptide (CNP), encoded by the natriuretic peptide precursor C (Nppc) gene, is an essential factor for maintaining oocyte meiotic arrest. In bovines, we have demonstrated an efficient and essential role of CNP in maintaining oocyte meiotic arrest. CNP derived from MGCs targets directly the NPR2 localized on both cumulus cells (CCs) and oocytes, and thus increase intra-oocyte cGMP levels via both CC-dependent and independent pathways. After that, increased intra-oocyte cGMP levels leads to an elevation of intra-oocyte cAMP levels by inhibiting PDE3A, and thus maintains oocyte meiotic arrest. This mechanism is very distinct from that reported in mice and porcines, in which only CC-dependent pathway functions via gap junctions (Zhang et al., 2010; Hiradate et al., 2014).
Based on the direct and efficient inhibitory effect on meiotic resumption in bovine oocytes, CNP could be a potential candidate for improving IVM method. Until the application of the invention, CNP, as a naturally presented factor in follicle fluid, has not been used for improving oocyte IVM methods.